RESEARCH ARTICLE MYOD1 functions as a clock amplifier as well as a critical co-factor for downstream circadian gene expression in muscle Brian A Hodge1†‡, Xiping Zhang1†, Miguel A Gutierrez-Monreal1, Yi Cao2, David W Hammers3, Zizhen Yao4, Christopher A Wolff1, Ping Du1, Denise Kemler1, Andrew R Judge5, Karyn A Esser1* 1Department of Physiology and Functional Genomics, University of Florida, Gainesville, United States; 2Department of Bioinformatics and Computational Biology, Genentech Inc, South San Francisco, United States; 3Department of Pharmacology and Therapeutics, University of Florida Health Science Center, Gainesville, United States; 4Allen Institute for Brain Science, Seattle, United States; 5Department of Physical Therapy, University of Florida Health Science Center, Gainesville, United States Abstract In the present study we show that the master myogenic regulatory factor, MYOD1, is a positive modulator of molecular clock amplitude and functions with the core clock factors for expression of clock-controlled genes in skeletal muscle. We demonstrate that MYOD1 directly regulates the expression and circadian amplitude of the positive core clock factor Bmal1. We identify a non-canonical E-box element in Bmal1 and demonstrate that is required for full MYOD1- responsiveness. Bimolecular fluorescence complementation assays demonstrate that MYOD1 *For correspondence: colocalizes with both BMAL1 and CLOCK throughout myonuclei. We demonstrate that MYOD1 and [email protected] BMAL1:CLOCK work in a synergistic fashion through a tandem E-box to regulate the expression † These authors contributed and amplitude of the muscle specific clock-controlled gene, Titin-cap (Tcap). In conclusion, these equally to this work findings reveal mechanistic roles for the muscle specific transcription factor MYOD1 in the Present address: ‡Buck Institute regulation of molecular clock amplitude as well as synergistic regulation of clock-controlled genes for Research on Aging, Novato, in skeletal muscle. United States DOI: https://doi.org/10.7554/eLife.43017.001 Competing interest: See page 21 Funding: See page 21 Introduction Received: 19 October 2018 Circadian rhythms are repetitive ~24 hr cycles that allow organisms to temporally align behavioral, Accepted: 20 February 2019 biochemical and physiological processes with daily environmental changes (Vitaterna et al., 2001; Published: 21 February 2019 Panda et al., 2002; Bhadra et al., 2017). These rhythms exist in virtually all mammalian cells and are generated by transcriptional/translational feedback loops referred to as the molecular-clock Reviewing editor: Andrew Brack, University of California, (Partch et al., 2014; Tataroglu and Emery, 2015; Takahashi, 2016). The positive limb of the core San Francisco, United States clock is comprised of the PAS domain containing basic-Helix-Loop-Helix factors (PAS-bHLH) core clock factors Brain and Muscle Arnt-Like 1 (Bmal1) and Circadian Locomotor Output Clocks Kaput Copyright Hodge et al. This (CLOCK). These factors heterodimerize and bind to the DNA at E-box elements where they generate article is distributed under the circadian transcription oscillations through rhythmic recruitment of histone acetylases, co-factors, terms of the Creative Commons Attribution License, which and components of the transcriptional complex (King et al., 1997; Bunger et al., 2000; permits unrestricted use and Partch et al., 2014). In addition to keeping time, the core molecular clock factors regulate the redistribution provided that the expression of downstream clock-controlled genes (CCGs), many of which encode master transcrip- original author and source are tional regulators and rate-limiting enzymes in key biochemical pathways (Bozek et al., 2007; credited. Bozek et al., 2009). Hodge et al. eLife 2019;8:e43017. DOI: https://doi.org/10.7554/eLife.43017 1 of 26 Research article Cell Biology Chromosomes and Gene Expression Although the core molecular clock components are expressed in the majority of cell-types throughout the body, CCGs are expressed in a very tissue-specific fashion (Storch et al., 2002; Zhang et al., 2014; Mure et al., 2018). This temporal regulation of tissue-specific gene programs allows for the timing of organ and cell-type specific processes that help maintain physiological homeostasis within each tissue and across multiple organ systems throughout the day (Bozek et al., 2009; Korencˇicˇ et al., 2015). The transcriptional mechanisms by which the core clock factors regu- late tissue-specific genes are not fully understood. Recent studies have begun to identify lineage- specific transcriptional regulators that co-localize with molecular clock components at cis-regulatory elements located within tissue-specific promoter and enhancer regions (Bozek et al., 2007; Dufour et al., 2011; Korencˇicˇ et al., 2012; Perelis et al., 2015). To date, factors within the liver, hippocampus, pancreas have previously been reported, however a muscle-specific transcriptional regulator has yet to be defined. In skeletal muscle the bHLH transcription factor MYOD1 drives myogenic gene expression by recruiting co-factors and the transcriptional machinery to muscle-specific gene promoters (Rudnicki et al., 1993; Polesskaya et al., 2001; Fong and Tapscott, 2013; Buckingham and Rigby, 2014). MYOD1 is often referred to as the ‘master myogenic switch’ as it is required for muscle cell differentiation and is capable of converting non-muscle cells into a muscle lineage (Davis et al., 1987; Tapscott et al., 1988). In adult skeletal muscle, BMAL1:CLOCK target the core-enhancer ele- ment (CE) located 20 kb upstream of the Myod1 start site to promote the circadian expression of MYOD1 (Andrews et al., 2010; Zhang et al., 2012). We previously reported that MYOD1-CE mice, that only lack the upstream CE region, display significant declines in the circadian amplitude of the core clock genes Bmal1 and Per2 (Zhang et al., 2012), suggesting MYOD1 may modulate core clock gene expression in skeletal muscle. Herein, we sought to address two questions: 1) Does MYOD1 transcriptionally regulate core molecular clock genes? And 2) Does MYOD1 work with the core clock components to regulate the circadian expression of muscle specific genes? We found that MYOD1 binds to an intronic enhancer within the Bmal1 promoter and functions to transcriptionally regulate Bmal1 expression. Using both In vivo and In vitro approaches we determined that MYOD1 serves to enhance the amplitude of Bmal1 expression creating a feed-forward regulatory loop between MyoD1 and the core clock gene, Bmal1 in skeletal muscle. We also found that MYOD1 works in a synergistic fashion with BMAL1: CLOCK to amplify the circadian expression of a muscle-specific, clock-controlled gene, Titin-cap (Tcap). Co-localization studies demonstrated that MYOD1, BMAL1, and CLOCK are in close proxim- ity within myonuclei. Tcap promoter analysis uncovered that MYOD1 and BMAL1 target a tandem E-box and that both Eboxes are required for the circadian regulation. These findings identify a novel role for MYOD1 as a clock amplifier and highlight synergistic interactions among core the clock fac- tors, BMAL1:CLOCK and MYOD1 in regulating downstream clock-controlled gene expression in skeletal muscle. Results Characterization of MYOD1 binding sites in adult skeletal muscle We first noted that expression of the core clock genes Bmal1 and Per2 were dampened in skeletal muscle of mice in which circadian expression of MyoD1 was abolished (MYOD1-CE mice), which sug- gested that MYOD1 may function as an upstream transcriptional regulator of the molecular clock (Zhang et al., 2012). To address these findings we performed a MYOD1 ChIP-Seq experiment with adult skeletal muscle from male C57BL/6J mice. We identified 12,343 MYOD1 binding sites on 7751 genes using very stringent statistics for calling peaks to minimize false positives due to our lack of a preimmune serum control (Supplementary file 1). We compared the list of genes bound by MYOD1 to a list of circadian genes identified from a high resolution time-series collection in skeletal muscle (Zhang et al., 2014). Of the 1454 circadian mRNA transcripts in skeletal muscle (JTK_CYCLE p-value < 0.03: Supplementary file 2) we found that approximately 30% (536 genes, Supplementary file 3) are directly targeted by MYOD1 (Figure 1A)(Zhang et al., 2014). Gene ontology (GO) enrichment analysis of these 536 circadian MYOD1 target genes revealed a significant enrichment for genes involved in muscle structure and development consistent with MYOD1’s known function as a myogenic transcription factor (Figure 1B, Supplementary file 4). Hodge et al. eLife 2019;8:e43017. DOI: https://doi.org/10.7554/eLife.43017 2 of 26 Research article Cell Biology Chromosomes and Gene Expression A B MYOD1 ChIP-Seq. MYOD1 bound circadian genes (7751 genes) muscle tissue development striated muscle tissue development circadian regulation 7215 536 918 of gene expression circadian rhythm Skeletal Muscle 0 2 4 6 8 10121416 Circadian Transcriptome - log p-value (1454 genes) CDE Asb2 mRNA Nrip1 mRNA Ppp1r3c mRNA sleveL 2.0 2.5 2.0 * WT WT MYOD1-CE * * 2.0 MYOD1-CE * * 1.5 * * 1.5 * A * N * 1.5 * R 1.0 * 1.0 m 1.0 evita * 0.5 0.5 WT 0.5 MYOD1-CE leR 0.0 0.0 0.0 18 22 26 30 34 38 42 18 22 26 30 34 38 42 18 22 26 30 34 38 42 CT CT CT F G Vegfa mRNA s l 6.0 BH.Q ADJ.P eve * WT Asb2 WT 0.0001 7.35E-05 5.0 MYOD1-CE L Abs2 MYOD1-CE 0.0455 0.04554 A 4.0 * Nrip1 WT 0.001 0.00049 N R 3.0 Nrip1 MYOD1-CE 0.1612 0.16123 m Ppp1r3c WT 0.001 0.00049 e 2.0 v it Ppp1r3c MYOD1-CE 0.0175 0.01745 1.0 a le Vegfa WT 0.0208 0.01039 R 0.0 Vegfa MYOD1-CE 1 1 18 22 26 30 34 38 42 CT Figure 1. MYOD1 binding coverage on skeletal muscle circadian genes. (A) Overlap of genes bound by MYOD1 (red) and circadian genes (grey) in adult skeletal muscle (JTK_CYCLE p-value < 0.03).